Relationship between the expression of MMP-2, TIMP-2 and VEGF-C in hepatocellular carcinoma with or without lymph node metastasis

AIM: To investigate the relationship between the expression of MMP-2, TIMP-2 and VEGF-C in hepatocellular carcinoma (HCC) with or without lymph node metastasis. METHODS: The expression of MMP-2, TIMP-2 and VEGF-C in 44 cases of HCC were examined using immunohistochemistry methods (SP).RESULTS: The positive expression of MMP-2, TIMP-2 and VEGF-C was associated with lymph node metastasis of HCC (P<0.05). CONCLUSIONS: There was a significant correlation between the high-expression of MMP-2 and VEGF-C, and the low-expression of TIMP-2 in lymph node metastasis of HCC. VEGF-C is an important factor promoting lymph node metastasis of HCC. The expression of MMP-2, TIMP-2 and VEGF-C may have important value in determining lymph node metastasis and prognosis of HCC.     

Effects of low-molecular weight heparin on MMP-2, TIMP-2 expression and invasiveness of cytotrophoblastic cells

AIM: To investigate the hypothesis that low-molecular weight heparin (LMWH) regulates in vitro cytotrophoblast invasiveness and production of metalloproteinases-2 (MMP-2), tissue inhibitor of metalloproteinas-2 (TIMP-2). METHODS: Chorionic villi tissue of normal 6-8 weeks pregnancy was obtained. Trophoblastic cells were collected by trypsin-collagenase digestion and Percoll gradient centrifugation. The cytotrophoblastic cells were cultured for 24 h and divided into 4 groups according to the concentrations (1.0×102 IU/L, 1.0×103 IU/L or 1.0×104 IU/L) of LMWH adding into the medium. The contents of MMP-2 and TIMP-2 in cell culture supernatants were measured by the method of ELISA. Cytotrophoblast invasiveness was determined by Transwell chamber assay. RESULTS: With the increasing concentrations of LMWH, the invasion activity of cytotrophoblastic cells and MMP-2 secretion were increased. At concentration of 1.0×103IU/L, LMWH greatly enhanced cytotrophoblast invasiveness and the expression of MMP-2 (P<0.05). The levels of TIMP-2 were decreased after intervention with LMWH. At concentration of 1.0×103IU/L or 1.0×104 IU/L, LMWH induced a significant decrease in TIMP-2 expression. No significant difference between group 1×103IU/L and group 1.0×104 IU/L was observed (P>0.05). CONCLUSION: LMWH might regulate cytotrophoblast invasiveness in vitro by influencing the expression of MMP-2 and TIMP-2 in cytotrophoblastic cells.     

Effects of chloroquine on autophagy and collagen metabolism in activated hepatic stellate cells in vitro

AIM: To explore the effects of chloroquine (CQ) on collagen Ⅰand collagen Ⅲ expression in activated rat hepatic stellate cell line HSC-T6 and the possible mechanism.METHODS: Transforming growth factor-β1 (TGF-β1) was used to activate HSC-T6 cells and 3 doses of CQ was administered for 24 h. The cells were divided into 5 groups as follows:control group, TGF-β1 group, TGF-β1+CQ (15 μmol/L) group, TGF-β1+CQ (30 μmol/L) group and TGF-β1 + CQ (60 μmol/L) group. Western blot was used to determine the expression of LC3-Ⅱ/LC3-I, P62 and α-SMA in activated HSC-T6 cells. The expression of collagen I and collagen Ⅲ was detected by immunocytochemical staining, Western blot and RT-qPCR. Western blot and RT-qPCR were also used to detect the expression of matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 at mRNA and protein levels.RESULTS: The ratio of LC3-Ⅱ/LC3-Ⅰ and P62 expression were increased after CQ intervention. Moreover, they were significantly higher in the TGF-β1+CQ groups than those in TGF-β1 group (P<0.01). The expression of collagen I and collagen Ⅲ at mRNA and protein levels was significantly increased in all TGF-β1+CQ groups as compared with TGF-β1 group (P<0.01), and it was markedly increased among TGF-β1+CQ groups in a dose-dependent manner. The expression of MMP-13 at mRNA and protein levels was significantly lowered and that of TIMP-1 and TIMP-2 was significantly increased in TGF-β1+CQ groups as compared with TGF-β1 group (P<0.05).CONCLUSION: Inhibition of autophagy by CQ in activated HSC-T6 cells up-regulates the expression of collagen I and collagen Ⅲ in a dose-dependent way, probably due to reduction of MMP-13 and enhancement of TIMP-1 and TIMP-2 expression.     

Roles of four liver cells in regulation of PA-plasmin system during liver fibrogenesis in rats

AIM: To investigate the roles of hepatocytes, hepatic stellate cells (HSCs), Kupffer cells (KCs) and endothelial cells (ECs) in the regulation of PA-plasmin system during liver fibrogenesis in rats. METHODS: Experimental liver fibrosis was induced in rats by injection of carbon tetrachloride (CCl₄) twice a week for 12 weeks. Four kinds of liver cells were separated from the normal and fibrotic livers of the rats. The expression levels of urokinase plasminogen activator (uPA), uPA receptor (uPAR) and PA inhibitor-1 (PAI-1) in liver cells were determined by Northern and Western blotting. RESULTS: The expression of PAI-1 and uPAR was markedly increased in HSCs during liver fibrosis in rats as compared to those in the ECs. CONCLUSION: HSCs and ECs may play very important roles in the regulation of PA-plasmin system during liver fibrogenesis in rats. The activated HSCs are main cells to secrete PAI-1 and uPAR.     

Changes of matrix metalloproteinase-3 and urokinase-type plasminogen activator receptor in type 2 diabetes mellitus with macroangiopathy

AIM: To investigate the function of matrix metalloproteinase-3 (MMP-3) and urokinase-type plasminogen activator receptor (uPAR) to macroangiopathy in type 2 diabetes mellitus. METHODS: The levels of MMP-3 and uPAR in plasma were determined by ELISA sandwich method in 26 healthy controls and 39 patients with type 2 diabetes mellitus including 15 complication-free cases and 24 with macroangiopathy. RESULTS: The plasma level of uPAR but not MMP-3 was higher in patients without macroangiopathy than that in normal controls (P<0.05). In patients with macroangiopathy, the plasma levels of MMP-3 and uPAR were higher than those in normal controls (P<0.01) and patients without angiopathy. There was positive relationship between MMP-3 and uPAR in type 2 diabetes mellitus (r=0.405, P<0.05), and LDL-C was the important affecting factor of uPAR. CONCLUSION: MMP-3 and uPAR are associated with the development of macroangiopathy in type 2 diabetes mellitus. MMP-3 can be regarded as the circulatory markers in type 2 diabetes mellitus with macroangiopathy.     

Oxysterols downregulate tissue inhibitor of metalloproteinase -1 expression in vascular smooth muscle cells

AIM:To investigate the effects of oxysterols on matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression in vascular smooth muscle cells. METHODS:Rabbit aortic vascular smooth muscle cells were culturedin vitroand incubated with cholesterol, Triol and 25-hydroxycholesterol (25-OH), respectively. Slot blot was used to detect the mRNA expression level of TIMP-1 in vascular smooth muscle cells (VSMCs); meanwhile the protein expression level of MMP-9 and TIMP-1 was detected by immunohistology. RESULTS:Triol and 25-OH inhibited the expression of TIMP-1 compared with control and cholesterol, but have no effect on expression of MMP-9. CONCLUSION:Both Triol and 25-OH downregulated TIMP-1 expression in VSMCs.     

Effects of IOX1 on proliferation,apoptosis and extracellular matrix-related protein expression in LX2 cells induced by TGF-β

AIM To investigate the effects of histone demethylase inhibitor IOX1 (5-carboxy-8-hydroxyquinoline) on the proliferation, apoptosis and extracellular matrix (ECM)-related protein expression in transforming growth factor-β (TGF-β)-induced human hepatic stellate LX2 cells. METHODS The proliferation and apoptosis of the LX2 cells were determined by real-time cell analysis and flow cytometry, respectively. The level of histone H3 lysine 9 dimethylation (H3K9me2) and the protein expression of ECM-related molecules [α-smooth muscle actin (α-SMA), collagen type I (Col I), matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1)] in the LX2 cells were detected by Western blot. RESULTS Treatment with IOX1 at 50~300 μmol/L significantly inhibited LX2 cell proliferation, and 300 μmol/L IOX1 significantly promoted the apoptosis of the LX2 cells. In addition, different concentrations of IOX1 increased the levels of H3K9me2 and MMP-1, and down-regulated the expression of α-SMA, Col I and TIMP-1 in TGF-β-induced LX2 cells (P<0.05). CONCLUSION Treatment with IOX1 inhibits the proliferation of LX2 cells induced by TGF-β, promotes the cell apoptosis, and regulates the synthesis and metabolism of ECM by elevating H3K9me2 level, thus attenuating hepatic fibrosis.     

Effects of genistein on proliferation,apoptosis and invasiveness in methotrexate-resistant human choriocarcinoma JAR/MTX cells and it s mechanism

AIM:To study the effects of genistein on JAR/MTX cell proliferation, apoptosis and invasion and it's mechanism in vitro. METHODS:MTT assay, Annexin-Ⅴ and propidium iodide label analysis and invasion assay were used to determine the effects of genistein on proliferation, apoptosis and invasiveness in JAR/MTX methotrexate- resistant human choriocarcinoma cells. RT-PCR was used to estimate the relative mRNA amounts of estrogen receptor(ER), MTA3 and snail in the cells. Western blotting and gelatin zymography assay were used to estimate the relative protein amounts of MMP-2, MMP-9 and E-cadherin in the cells. RESULTS:After treatment of genistein, the proliferation and invasiveness of JAR/MTX cells were decreased significantly in a dose-dependent manner. 10 μmol/L genistein induced apoptosis, whereas 25, 50, 100 μmol/L genistein induced apoptosis and necrosis significantly. Genistein led to an increase in ERβ, MTA3 mRNA and E-cadherin protein expression, and decreases in the amounts for snail mRNA and MMP-2 and MMP-9 protein expression of JAR/MTX cells. CONCLUSIONS:Genistein inhibits the cell proliferation by inducing cell apoptosis and necrosis. Genistein also may inhibit JAR/MTX cell invasion in part through the upregulation of E-cadherin and downregulation of MMP-2 and MMP-9. The signal transduction pathway of invasion suppression induced by genistein in JAR/MTX cells may be as follows: MTA3→snail→ E-cadherin.     

Expression of matrix metalloproteinase and tissue inhibitor of metalloproteinase in lung tissue of hypercapnia rat model

AIM: To study the expression of matrix metalloproteinase-9(MMP-9), matrix metalloproteinase-2(MMP-2) and the tissue inhibitor of metalloproteinase(TIMP-1) in the lung tissue of the hypercapnia rat.METHODS: Forty Wistar rats were randomly divided into a control group (group A, n=20) and hypercapnia group (group B, n=20). Group B received mix gas exposure (6% CO2, 21% O2, 72% N2) 7 h daily for 4 weeks. The parameters we would examine were as follow: arterial blood gas; the mean pulmonary artery pressure;MMP-2,MMP-9, TIMP-1, and NE activity in lung tissue. Masson pigmentation of elasticity fibre was analyzed by computer image analyzer. Histopathological changes of lung tissue were observed under light microscope. The protein expression of MMP (MMP-2, MMP-9) and TIMP (TIMP-1) in lung tissue were determined by immunocytochemistry.RESULTS: Decompensate respiratory acidosis (pH=7.20±0.04, PaCO2=7.84±0.15) developed in group B. The mean pulmonary artery pressure were similar between groups B and A (P>0.05). Tissue edema in the lung, endothelial cell damage of the small blood vessels, pulmonary micro thrombus formations and increased pulmonary capillary permeability were observed in group B. NE activity increased significantly (P<0.01). However, no significant change of MMP-2, MMP-9, TIMP-1 activity was found in group B and group A (P>0.05). There was significant decrease in the relative content of elasticity fibre in lung tissue in group B compared to group A (P<0.01). The expression of MMP-2 protein in the lung tissue of group B was lower than that in group A (P<0.01), but the expression of both MMP-9 and TIMP-1 proteins in the lung tissue in group B were higher than those in group A (P<0.01).CONCLUSION: Hypercapnia rat model is successfully reproduced by exposure of animals to the mix gas exposure (6% CO2, 21% O2, and 72% N2). The pulmonary artery pressure is not affected by hypercapnia. High concentration of CO2 causes increase of NE activity and decrease in the relative content of elasticity fibre. High concentration of CO2 causes the increase of MMP-2 protein expression and decrease in the MMP-9 and TIMP-1 protein expression.     

Effect of long non-coding RNA MEG3 on invasion and migration of colorectal cancer cells

AIM: To investigate the expression of long non-coding RNA maternally expressed gene 3(MEG3) in colorectal cancer(CRC) cells, and to observe the effect of MEG3 on the invasion and migration of CRC cells. METHODS: The levels of MEG3 in human normal colon cell NCM460 and CRC cells SW48 and LoVo were detected by real- time PCR. MEG3 was over-expressed by plasmid transfection, and the effects of MEG3 on the invasion and migration of SW48 and LoVo cells were analyzed by Transwell assay and wound healing assay. The expression of matrix metalloproteinase(MMP) family proteins was determined by Western blotting. RESULTS: The level of MEG3 was down-regulated in CRC cells compared with normal colon cell NCM460. The invasion and migration of CRC cells were reduced after MEG3 over-expression. Transwell invasion and migration assays showed that the numbers of transmembrane SW48 and LoVo cells were smaller in MEG3 over-expression group than control group(CONCLUSION: The expression of MEG3 is down-regulated in CRC cells. Over-expression of MEG3 inhibits the invasion and migration of CRC cells. TIMP-2, MMP-2 and MMP-9 might play an important role in this regulation.     

Overexpression of N-terminal amino acids of p55γ regulatory subunit of PI3K inhibits cell adhesion of human gastric carcinoma MGC803 cells

AIM: To investigate the effect of the N-terminal 24-amino acid(N24) overexpression in p55γ re-gulatory subunit of phosphatidylinositiol 3-kinase(PI3K) on the cell adhesion of human gastric carcinoma cell MGC803.METHODS: MGC803 cells, which stably expressed GFP-N24 fusion protein and GFP alone, were generated by transfection with pEGFPN-24 plasmid and control plasmid pEGFP-C1, respectively. The morphological change of the cells was observed under inverted microscope. The expression of GFP-N24 fusion protein was detected by Western blot. The adhesion of the cells was determined by cell adhesion assay. The effects of GFP-N24 fusion protein on the expression of E-cadherin and β-catenin were analyzed by Western blot. The expression and secretion of matrix metalloproteinase 9(MMP9) and urokinase-type plasminogen activator(uPA) in the MGC803 cells were detected by gelatin zymography.RESULTS: MGC803/GFP-N24 cell line steadily expressed GFP-N24 fusion protein and MGC803/GFP cell line steadily expressing GFP were successfully established, but the expression of fusion protein GFP-N24 was lower than that of the control protein GFP. The morphological changes of the transfected cells from paving stone to fibroblast cell form after gene transfection, and the cytoplasm secretory granules were increased significantly. The cell adhesion to fibronectin and collagen decreased after GFP-N24 transfection. GFP-N24 fusion protein increased the expression of cell adhesion molecule E-cadherin and decreased the wnt signal pathway molecule β-catenin in the MGC803 cells. However, it did not affect the expression and secretion of tumor metastasis-related proteins MMP9 and uPA.CONCLUSION: Overexpression of N24p55γ inhibits cell adhesion by influencing the expression of E-cadherin and β-catenin in the MGC803 cells.     

Detection and analyses of coagulation and fibrolysis factor mRNA levels in tissue of hepatocellular carcinoma patients

AIM:To detect the mRNA expression of tissue factor (TF), urokinase-type plasminogen activator (u-PA) and urokinase-type plasminogen activator receptor (u-PAR) in the samples of hepatocellular carcinoma (HCC) and adjacent tissue, and to elucidate their association with clinical significance. METHODS:The mRNA levels of TF, u-PA and u-PAR in 27 human HCC tissue samples with their adjacent tissue samples and 27 normal liver tissues were detected by RT-PCR. The relationship between the mRNA expression and their clinic-pathological data were also analyzed. RESULTS:The expressive rate of TF, uPA and uPAR in HCC tissue samples was 62.96% (17/27), 70.37% (19/27), 77.78% (21/27), respectively and relative expression intensity of TF, uPA and uPAR were 0.567±0.268, 0.964±0.458 and 0.784±0.322, respectively, which were significantly higher than those in adjacent tissue group and normal liver group (P<0.05). TF, uPA and uPAR mRNA levels were all significantly higher in larger size and in the invasive and metastatic subgroup (P<0.05). There had positive relationship between the mRNA expressions of these three factors. CONCLUSION:The results indicate that TF, uPA and uPAR might play an important role in hepatic carcinogenesis, invasiveness and metastasis.     

MMP-7,MMP-10 and TIMP-4 expression in ventricular remodeling of human heart failure

AIM:To sieve matrix metalloproteinases (MMPs) and the tissue inhibitors of matrix metalloproteinases (TIMPs) closely associated with ventricular remodeling of human heart failure using antibody chip technology.METHODS:We performed cytokine-specific antibody array analysis using individual left ventricular myocardial samples from 6 patients with heart failure due to arrythmogenic right ventricular cardiomyopathy (ARVC) undergoing transplantation and matched samples from 6 non-failing subjects to screen differentially expressed MMPs and TIMPs associated with the ventricular remodeling of heart failure.The results were further validated by ELISA and immunohistochemical analysis.RESULTS:We identified high expression of MMP-7 and MMP-10 and low expression of TIMP-4 in ARVC failing hearts compared to non-failing hearts by hybridization with the cytokine-specific antibody arrays containing 17 MMPs and 4 TIMPs on the chips.ELISA and immunohistochemical analyses further confirmed that differentially expressed levels of MMP-7, MMP-10, and TIMP-4 were observed not only in ARVC failing heart, but also in failing hearts due to ischemic (ICM) and dilated cardiomyopathy (DCM).CONCLUSION:Highly expressed MMP-7 and MMP-10 and lowly expressed TIMP-4 may be involved in the ventricular remodeling of heart failure derived from cardiomyopathy of different etiology.     

miR-496 over-expression inhibits growth and metastasis in colon cancer cells

AIM: To investigate the effect of miR-496 over-expression on the growth and metastasis of colon cancer cells and its molecular mechanism.METHODS: The proteins interacting with miR-496 were screened by bioinformatic method. The levels of miR-496, CTNNB1 mRNA and β-catenin protein in colon cancer cell lines, HT29, HCT116 and SW480, and normal colonic epithelial cell line NCM460 were detected by real-time PCR and Western blot. HT29, HCT116 and SW480 cells were transfected with miR-496 mimics using Lipofectamine 2000 and named as HT29-miR-496 mimics, HCT116-miR-496 mimics and SW480-miR-496 mimics cells, respectively, and the cells transfected with the scramble served as negative control. The cell viability, lactate dehydrogenase (LDH) leakage, and colony formation and metastatic abilities were determined by MTT assay, LDH assay, colony formation assay and Transwell method, respectively. The promoter activity of miR-496 was measured using luciferase reporter gene assay. The protein levels of β-catenin, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), p-4E-BP1, low-density lipoprotein receptor-related protein 6(LRP6), p-LRP6, MMP-7, MMP-9, MMP-13 and TIMP-2 were monitored by Western blot.RESULTS: Endogenous miR-406 interacted with β-catenin was found in the colon cancer cells. Low miR-496 expression in the HT29, HCT116 and SW480 cells and high miR-496 expression in NCM460 cells were detected. In contrast, high β-catenin expression was found in the HT29, HCT116 and SW480 cells and low β-catenin expression was observed in the NCM460 cells. Compared with control group, the cell viability, colony formation rate and the number of metastatic cells remarkably decreased in the HT29-miR-496 mimics, HCT116-miR-496 mimics and SW480-miR-496 mimic cells (P<0.05). The promoter activity of miR-496 was significantly increased in colon cancer cells transfected with miR-496 mimics, and was 1.75, 2.04 and 1.61 times as high as control group. miR-496 over-expression inhibited β-catenin levels, and p-4E-BP1 and p-LRP6 protein levels were also reduced. siRNA- or over-expressed miR-496-mediated β-catenin down-regulation inhibited MMP-7 and MMP-9 expression, but promoted TIMP-2 expression.CONCLUSION: The expression level of miR-496 in the colon cancer cells is low, but in the normal colonic epithelial cells is high. miR-496 over-expression inhibits the protein levels of MMP-7 and MMP-9, and promotes the protein expression of TIMP-2 via inhibiting Wnt/β-catenin pathway, thus suppressing malignant phenotype in the colon cancer cells.     

Mechanism of miR-30c over-expression inhibiting malignant phenotypes of cervical cancer cells

AIM:To investigate the molecule mechanism of microRNA (miR)-30c over-expression inhibiting malignant phenotypes of cervical cancer cells. METHODS:Cervical cancer cell lines C33A, HeLa, SiHa and CaSki were transfected with pGenesil-1-miR-30c plasmid using Lipofectamine 2000 kit, and the expression of miR-30c was determined by TaqMan real-time PCR. The cell viability inhibition rate, colony formation ability, migration rate and apoptotic rate were measured by MTT assay, colony formation assay, Transwell experiment, and flow cytometry with Annexin V-FITC staining. The protein expression of Bax, Bcl-2, matrix metalloproteinase (MMP)-9, MMP-13 and tissue inhibitor of metalloprotei-nase-1 (TIMP-1) was detected by Western blot. RESULTS:The expression levels of miRNA-30c in the cervical cancer cell lines transfected with pGenesil-1-miR-30c plasmid were significantly higher than those in negative control groups (cell lines transfected with pGenesil-1 plasmid) (P<0.01). Significantly increased cell viability inhibition rate, and decreased colony formation ability and migration rate were found in the cervical cancer cell lines over-expressing miR-30c as compared with negative control groups (P<0.05). The apoptotic rate in the cervical cancer cell lines over-expressing miR-30c was dramatically higher than that in control groups (P<0.05). Over-expression of miR-30c in cervical cancer cells promoted the protein expression of Bax and TIMP-1, and decreased the protein expression of Bcl-2 and MMP-13 (P<0.05 or P<0.01). CONCLUSION:Over-expression of miR-30c significantly inhibits the viability and migration, and induces apoptosis of cervical cancer cells. The mechanism may be related to activating apoptosis pathway and inhibiting MMP-13 protein expression.